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Insight into the sequence specificity of a probe on an Affymetrix GeneChip by titration experiments using only one oligonucleotide

View Article: PubMed Central - PubMed

ABSTRACT

High-density oligonucleotide arrays are powerful tools for the analysis of genome-wide expression of genes and for genome-wide screens of genetic variation in living organisms. One of the critical problems in high-density oligonucleotide arrays is how to identify the actual amounts of a transcript due to noise and cross-hybridization involved in the observed signal intensities. Although mismatch (MM) probes are spotted on Affymetrix GeneChips to evaluate the noise and cross-hybridization embedded in perfect match (PM) probes, the behavior of probe-level signal intensities remains unclear. In the present study, we hybridized only one complement 25-mer oligonucleotide to characterize the behavior of duplex formation between target and probe in the complete absence of cross-hybridization. Titration experiments using only one oligonucleotide demonstrated that a substantial amount of intact target was hybridized not only to the PM but also the MM probe and that duplex formation between intact target and MM probe was efficiently reduced by increasing the stringency of hybridization conditions and shortening probe length. In addition, we discuss the correlation between potential for secondary structure of target oligonucleotide and hybridization intensity. These findings will be useful for the development of genome-wide analysis of gene expression and genetic variations by optimization of hybridization and probe conditions.

No MeSH data available.


Absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04 with cDNA background. (A) PM, MM signals, and PM/MM ratios, which were demonstrated by the titration assay of hybridization with target oligonucleotide Dap3-02, with cDNA background. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses. (B) Effects of shortening target length on absolute signal intensities with cDNA background. The line plots show absolute signal intensities of PM and MM probes, which were increased by titration assay of hybridization with target oligonucleotide, Dap3-02-6nt. The bar graphs show the ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities were determined as described above.
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f4-3_47: Absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04 with cDNA background. (A) PM, MM signals, and PM/MM ratios, which were demonstrated by the titration assay of hybridization with target oligonucleotide Dap3-02, with cDNA background. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses. (B) Effects of shortening target length on absolute signal intensities with cDNA background. The line plots show absolute signal intensities of PM and MM probes, which were increased by titration assay of hybridization with target oligonucleotide, Dap3-02-6nt. The bar graphs show the ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities were determined as described above.

Mentions: Our findings that optimization of probe length and target length had a marked impact on specificity of duplex formation in the complete absence of cross-hybridization. These conditions were very different from the standard conditions of genome-wide analyses of gene expression and genetic variation. To validate our findings under standard hybridization conditions, we hybridized the target oligonucleotide in the presence of the complex cDNA background. Figures 4A and 4B show the absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04, that were increased by titration assay of hybridization with target oligonucleotides Dap3-02 and Dap3-02-6nt, respectively, with the cDNA background. As shown in Figure 1A, the cDNA background concealed the behavior of target-probe hybridization at less than or equal to a target concentration of 140 fM, and we therefore focused on the signal intensities obtained from the titration experiments above the target concentration of 1.4 pM. In the case of both Dap3-02 and Dap3-02-6nt, similar results were obtained in the presence of cDNA background. However, the addition of cDNA background reduced the PM/MM ratios slightly, although the absolute signal intensities of both PM and MM probes were decreased. It is expected that the addition of cDNA background would increase the absolute signal intensities, and the absolute signal intensities at the low target concentrations increased markedly. These results indicated that the effective target concentrations were reduced by target-target interactions between the target oligonucleotide and cDNA background in the hybridization solution.


Insight into the sequence specificity of a probe on an Affymetrix GeneChip by titration experiments using only one oligonucleotide
Absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04 with cDNA background. (A) PM, MM signals, and PM/MM ratios, which were demonstrated by the titration assay of hybridization with target oligonucleotide Dap3-02, with cDNA background. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses. (B) Effects of shortening target length on absolute signal intensities with cDNA background. The line plots show absolute signal intensities of PM and MM probes, which were increased by titration assay of hybridization with target oligonucleotide, Dap3-02-6nt. The bar graphs show the ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities were determined as described above.
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f4-3_47: Absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04 with cDNA background. (A) PM, MM signals, and PM/MM ratios, which were demonstrated by the titration assay of hybridization with target oligonucleotide Dap3-02, with cDNA background. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses. (B) Effects of shortening target length on absolute signal intensities with cDNA background. The line plots show absolute signal intensities of PM and MM probes, which were increased by titration assay of hybridization with target oligonucleotide, Dap3-02-6nt. The bar graphs show the ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities were determined as described above.
Mentions: Our findings that optimization of probe length and target length had a marked impact on specificity of duplex formation in the complete absence of cross-hybridization. These conditions were very different from the standard conditions of genome-wide analyses of gene expression and genetic variation. To validate our findings under standard hybridization conditions, we hybridized the target oligonucleotide in the presence of the complex cDNA background. Figures 4A and 4B show the absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04, that were increased by titration assay of hybridization with target oligonucleotides Dap3-02 and Dap3-02-6nt, respectively, with the cDNA background. As shown in Figure 1A, the cDNA background concealed the behavior of target-probe hybridization at less than or equal to a target concentration of 140 fM, and we therefore focused on the signal intensities obtained from the titration experiments above the target concentration of 1.4 pM. In the case of both Dap3-02 and Dap3-02-6nt, similar results were obtained in the presence of cDNA background. However, the addition of cDNA background reduced the PM/MM ratios slightly, although the absolute signal intensities of both PM and MM probes were decreased. It is expected that the addition of cDNA background would increase the absolute signal intensities, and the absolute signal intensities at the low target concentrations increased markedly. These results indicated that the effective target concentrations were reduced by target-target interactions between the target oligonucleotide and cDNA background in the hybridization solution.

View Article: PubMed Central - PubMed

ABSTRACT

High-density oligonucleotide arrays are powerful tools for the analysis of genome-wide expression of genes and for genome-wide screens of genetic variation in living organisms. One of the critical problems in high-density oligonucleotide arrays is how to identify the actual amounts of a transcript due to noise and cross-hybridization involved in the observed signal intensities. Although mismatch (MM) probes are spotted on Affymetrix GeneChips to evaluate the noise and cross-hybridization embedded in perfect match (PM) probes, the behavior of probe-level signal intensities remains unclear. In the present study, we hybridized only one complement 25-mer oligonucleotide to characterize the behavior of duplex formation between target and probe in the complete absence of cross-hybridization. Titration experiments using only one oligonucleotide demonstrated that a substantial amount of intact target was hybridized not only to the PM but also the MM probe and that duplex formation between intact target and MM probe was efficiently reduced by increasing the stringency of hybridization conditions and shortening probe length. In addition, we discuss the correlation between potential for secondary structure of target oligonucleotide and hybridization intensity. These findings will be useful for the development of genome-wide analysis of gene expression and genetic variations by optimization of hybridization and probe conditions.

No MeSH data available.